Image-based and biochemical assays to investigate endosomal protein sorting

Methods Enzymol. 2014;534:155-78. doi: 10.1016/B978-0-12-397926-1.00009-3.

Abstract

The sorting of membrane proteins within the endosomal system occurs through a panoply of highly dynamic sequential molecular interactions that together govern many physiologically important processes. A key component of the endosomal protein sorting machinery is the retromer complex. Through two distinct subcomplexes, retromer operates to select cargo for endosome-to-Golgi retrieval and also drives membrane tubule formation. Many accessory proteins associate with retromer to facilitate protein sorting and/or tubule formation. The experience we have gained from studying retromer-mediated endosomal protein sorting and the assays developed and applied in the course of these studies can provide a template for researchers interested in related endosomal trafficking pathways. Herein we describe image-based assays that can be applied to study endosomal protein sorting through the use of antibody-uptake assays in low-, medium-, and high-throughput formats. We additionally detail simple but effective native immunoprecipitation methods that can be employed to identify novel proteins that may interact transiently with a protein of interest within the endosomal pathway.

Keywords: Antibody-uptake assay; Endosome; Native immunoprecipitation; Quantitative imaging; Retromer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / chemistry
  • CD8 Antigens / genetics
  • CD8 Antigens / metabolism
  • Endoplasmic Reticulum / metabolism*
  • Endosomes / metabolism*
  • Gene Expression Regulation
  • Genes, Reporter
  • Golgi Apparatus / metabolism*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Immunoprecipitation
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Microscopy, Fluorescence
  • Protein Transport
  • Receptor, IGF Type 2 / genetics
  • Receptor, IGF Type 2 / metabolism
  • Signal Transduction
  • Sorting Nexins / genetics
  • Sorting Nexins / metabolism
  • Vesicular Transport Proteins / genetics
  • Vesicular Transport Proteins / metabolism

Substances

  • Antibodies, Monoclonal
  • CD8 Antigens
  • GOLPH3 protein, human
  • Membrane Proteins
  • Receptor, IGF Type 2
  • SNX1 protein, human
  • Sorting Nexins
  • VPS26A protein, human
  • Vesicular Transport Proteins
  • Green Fluorescent Proteins